Abstract
Overexpression of mutant p53 has been reported to promote tumorigenicity in several cancers. However, despite its potential importance, the signals regulating mutant p53 protein expression are not known. Here we show that a form of p53 that is incapable of binding DNA is overexpressed in the acute promyelocytic leukemia NB4 cell line. Our results demonstrate that treatment of NB4 cells with bryostatin-1, which induces differentiation in this cell line, leads to hyperphosphorylation of this DNA binding-impaired form of p53 via mitogen-activated protein kinase. After this phosphorylation, the p53 protein is degraded by the ubiquitin/proteasome pathway. Furthermore, we show that inhibition of p53 hyperphosphorylation blocks p53 protein degradation and cell differentiation. In addition, inhibition of the ubiquitin/proteasome pathway also blocks p53 protein degradation and cell differentiation. These findings suggest a role for mitogen-activated protein kinase in the degradation of the DNA binding-impaired form of p53 protein and in the bryostatin-induced differentiation observed in this cell line. The implications of these results with respect to the functional significance of p53 phosphorylation and degradation in cell differentiation are discussed.
Highlights
An important mechanism used to control p53 activity is the regulation of p53 protein levels
We demonstrate that inhibition of p53 hyperphosphorylation by a mitogen-activated protein kinase (MAPK) pathway inhibitor, PD98059, blocks p53 protein degradation and cell differentiation
MAPK Pathway Is Involved in p53 Hyperphosphorylation and Reduction in p53 Protein Levels—We have shown that MAPK is activated in NB4 cells after bryostatin treatment,2 which raised the possibility that the MAPK pathway might be involved in p53 hyperphosphorylation and protein reduction
Summary
Cell Culture and Protein Purification—NB4 cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 media supplemented with 10% fetal bovine serum. Detection of p53 Phosphorylation and Protein Level in NB4 Cells— Two ml of NB4 cells at 106 cell/ml were labeled with [32P]orthophosphate (ICN) in phosphate-deficient Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) and incubated for 1 h followed immediately by bryostatin-1 treatment. To determine p53 protein levels, NB4 cells were lysed with radioimmune precipitation buffer as described above, and the protein concentration for each sample was measured. Equivalent amounts of cell lysate were analyzed by SDS-PAGE followed by Western blot analysis using Pab 421. Supernatant containing an equivalent amount of protein from each sample was incubated with 20 l of packed agarose beads coupled to a monoclonal anti-phosphotyrosine antibody (PT-66, Sigma) at 4 °C overnight. The immunoprecipitate was analyzed by SDS-PAGE followed by Western analysis with a rabbit anti-p42 MAPK polyclonal antibody (Pab C14, Santa Cruz). Reactions were incubated for 30 min at 30 °C and analyzed on a 3% polyacrylamide gel containing 0.5 ϫ TBE
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